Shreds of biopsied adult human jaw bones were divided into four groups that were each incubated together with borosilicate glass chips in one of four variants of DMEM expansion medium (i.e., with a high or low calcium complement and with or without sodium ascorbate added). The outgrown bone cells were propagated in secondary cultures kept in the same DMEM expansion medium variants prior to being transferred to a beta-glycerophosphate-enriched high-Ca2+ and ascorbate-containing DMEM mineralisation medium. In this latter medium the proliferation rates and the simultaneous expression of alkaline phosphatase by the four distinct groups of osteoblasts were assessed during 7 days of staying in vitro via biochemical methods. Human osteoblasts previously expanded in high-Ca2+ ascorbate-added DMEM medium were found to initially express a quite high alkaline phosphatase activity that subsequently declined, while their proliferative activity remained rather low. Conversely, osteoblasts formerly expanded in low-Ca2+ ascorbate-devoid DMEM medium exhibited minimal levels of alkaline phosphatase activity while simultaneously maximally proliferating in the mineralisation medium. Moreover, a mixture (1:1 v/v) of the DMEM mineralisation medium with Ham's medium (in which the aminoacid proline abounds) was found to accelerate, in comparison to DMEM alone, the intracellular type I procollagen synthesis, the extracellular assembly of type I collagen fibrils, and the calcification of the extracellular matrix by the human osteoblasts. Hence, the presence or absence of calcium and/or ascorbate in the expansion medium and of proline in mineralisation medium can significantly modulate the progression of human jaw bone cells from an undifferentiated highly proliferating condition to the mature osteoblastic phenotype.